Optimized Protocols to Extract Total Transcripts and Proteins from Lipid-Rich Tissues.

BACKGROUND

Highly lipidic tissues (e.g., adipose tissue, brain, and liver) are challenging for transcript and protein extraction and for next-generation sequencing. Lipids can clog filters, columns, and pipettes; cause autofluorescence and quenching in imaging; and interfere with centrifugation-based separation.

AIM: To identify the most suitable method for extracting total RNA for RT-qPCR and an alternative method for extracting total protein for quantification in mice fed a regular or high-fat diet.

METHODS

We compared three total RNA extraction methods and two total protein extraction methods.

RESULTS

The highest total RNA yield and purity were obtained with TRIzol and chloroform, with optimized steps added to the original protocol to address the challenges posed by highly lipid-rich tissues. For total protein extraction, an adipose tissue-specific kit from Invent Biotechnologies yielded higher protein levels than the classical RIPA-based method. Among lipid-rich tissues, we observed that adipose tissue was more challenging to process than the brain and the liver.

CONCLUSIONS

Adipose tissue, particularly under a high-fat diet, is the most challenging lipid-rich tissue, followed by the brain and then the liver. We highlight protocols that improve total RNA and protein yields and purity, which may benefit other researchers working with these tissues.